Untangling the systematic dilemma behind the roughskin spurdog Cirrhigaleus asper (Merrett, 1973) (Chondrichthyes: Squaliformes), with phylogeny of Squalidae and a key to Cirrhigaleus species

Cirrhigaleus comprises a small genus of rare barbel-bearing dogfish sharks with distributions in limited regions of all oceans. Generic validity and taxonomic status of some species are upon controversies by morphological and molecular evidence that often suggest reallocation of Cirrhigaleus species into the genus Squalus. Particularly, the roughskin spurdog C. asper exhibits intermediary morphological characteristics within Squalidae that requires clarification. In the present study, a phylogenetic approach was undertaken to test the correct generic placement of C. asper using novel and revised morphological characters. We performed maximum parsimony analysis of 51 morphological characters of the internal (e.g., neurocranium, clasper cartilages, pectoral and pelvic girdles) and external anatomy applied to 13 terminal taxa. Cirrhigaleus represents a valid genus and it is supported by eight synapomorphies: high number of monospondylous vertebrae; medial nasal lobe supported by fleshy core and innervated by the buccopharyngeal branch of the facial nerve; neurocranium with greatest width across nasal capsules; one facet and one condyle in the puboischiadic bar for articulating with the basipterygium; two intermediate segments between the basipterygium of the pelvic fin and the axial cartilage of the claspers; five terminal clasper cartilages; and posterior medial process of the puboischiadic bar absent. Cirrhigaleus asper is sister-species to a small clade comprising C. barbifer and C. australis which is supported by one synapomorphy, presence of conspicuous cusplets in the dermal denticles. Cirrhigaleus barbifer, C. asper and C. australis are redescribed herein and the neotype of C. barbifer is designated. A key to Cirrhigaleus species is also given and the inner relationships within Squalus is tentatively discussed.

Introduction follows [3]. Cranial measurements were obtained from dissected specimens and follow [36,37] with modifications as in [35]. Subethmoidal ridge length is defined here as the longitudinal distance between its anteriormost edge at the rostral carina and its posteriormost edge between the subnasal fenestrae. External and cranial measurements are expressed, respectively, as percentages of total length (%TL) and of the neurocranium total length (%CL). In tables, mean and standard deviation are given to include all specimens in which data were taken, except for C. asper whose range values of measurements taken from fresh specimens are given separately.
Phylogenetic analysis was performed under parsimony using TNT 1.1 [53,54] and the 'Traditional Search' option, using the TBR (Tree Bisection Reconnection) algorithm. Quantitative (1)(2)(3)(4) and multistate qualitative (15, 30, 32 and 51) characters were analyzed as ordered. Missing entries were coded as '?' and used when appropriate study material was unavailable or character state was inapplicable for a given character. We used implied weighting herein and tested three different values of concavity constant, k (1, 3 and 5). CI (consistency index) and RI (retention index) values and synapomorphies of nodes were obtained from the set of equally most-parsimonious trees. The relative degree of support for each node in the trees obtained with implied weights was assessed with branch support indices [55] and symmetric resampling [53]. Relative Bremer support was calculated using TBR and retaining suboptimal trees by seven steps. GC values (differences of frequencies "Group present/Contradicted" [53]) were calculated using the strict consensus and 2000 replicates. Tree edition was performed with the aid of FigTree v1.4.3 and Adobe Photoshop CS6.
The statement of each character and its states is followed by its CI and RI. A list of synapomorphies is given below and begins in Squalidae and progressively continues to less inclusive clades within the family. For each clade, only non-ambiguous synapomorphies are listed (see S1 File). After each synapomorphy, the number of the referred character and its state changes are shown in brackets. A complete list of character transformations is presented in S2 File. Complete data matrix including quantitative and qualitative characters analyzed are given in S3 File and S4 File.

Taxonomic validation
Species recognized herein are redescribed under the section 'Taxonomic account'. Synonyms for species include authorship, date, pages and figures whenever possible. Diagnosis and key to species are based on adult specimens only (unless otherwise stated in text) due to ontogenetic and regional plasticity of phenotypic characteristics. In the descriptions, single values for morphometric and meristic data are for holotype whereas ranges represent values for all material in which data were taken (except when otherwise mentioned in text). Monospondylous vertebrae are defined as those vertebrae whose centrum corresponds to a single intermuscular septum and a pair of neural spines. Usually, these vertebrae are located between the occipital centrum in the neurocranium to over or posterior to the pelvic girdle [58]. This character has been used to separate taxonomically species of sharks as in [34] and not less obviously in Squalidae (e.g., [59]). In Dalatias and Isistius, total counts of monospondylous vertebrae vary from 37 (minimum) to 44 (maximum). In Squalus species, it varies from 37 (minimum) to 50 (maximum) whereas in Cirrhigaleus species it is 47-53.
Diplospondylous vertebrae are defined as those vertebrae whose centrum corresponds to two subsequent intermuscular septa and a pair of neural spines. These vertebrae are located from the pelvic girdle to the caudal tip [58]. In Dalatias and Isistius, total counts of diplospondylous vertebrae vary from 35 (minimum) to 47 (maximum). In Squalus species, it varies from 60 (minimum) to 77 (maximum) whereas in Cirrhigaleus species the range from 60 to 69. Total number of tooth rows in the upper and lower jaws are often expressed as a dental formula in taxonomic descriptions. These formulae vary widely within Squaliformes as seen in [60] and rarely are applied in phylogenetic analysis of sharks (e.g., [61,62]). In Dalatias and Isistius, upper tooth rows range from 16 (minimum) to 39 (maximum) and lower tooth rows varies from 15 (minimum) to 31 (maximum). In Squalus species, the range is 21-28 for upper tooth and 17-25 for lower tooth rows. Cirrhigaleus species show a minimum of 23 and maximum of 30 upper tooth rows and 18-27 for lower tooth rows.
Neurocranium and associated nerves.
In Cirrhigaleus, anterior margin of nostrils has a medial nasal lobe that bears fleshy core and it is innervated by the buccopharyngeal branch of facial nerve (VII) (5,1). The nerve buccopharyngeal branch of facial nerve (VII) arise from the foramen prooticum (V-VII) at the interorbital wall and runs aside the suborbital shelf of the neurocranium and over suborbitalis muscle in the palatoquadrate till it reaches the antorbital cartilage (Fig 1). Then, the branch buccopharyngeal runs ventrally in the subethmoidal region and reaches the nasal capsule in the neurocranium where it splits into two different small branches. The left branch is thicker than the right one and it runs to the origin of the nasal barbel (medial nasal lobe) at the fleshy core. The second branch runs straight to the rostral tip and aside the rostral keel. In S. acanthias, S. suckleyi, S. megalops, S. brevirostris, S. albifrons, S. mitsukurii, S. montalbani and S. japonicus, buccopharyngeal nerve runs exclusively to the tip of the rostrum with small innervations running to the nasal capsules, although these do not reach the anterior margin of nostrils. Thus, anterior margin of nostrils is not innervated in these species and the medial nasal lobe is internally supported by a thin nasal cartilage only, lacking fleshy core. In Dalatias, Isistius and species of Squalus such innervation is absent (5,0) [22]. Proposed this character as a synapomorphy of Cirrhigaleus. 6) Fleshy core at the anterior margin of nostrils: character state 0 -absent; character state 1present (CI = 100; RI = 100).
Species of Squalus present a paired short and cylindrical process located at the dorsoposterior edge of the prefrontal fontanelle in the neurocranium (7,1) (Fig 2C-2E; Fig 3C-3F). These processes are absent in Dalatias, Isistius and in both C. barbifer and C. australis (7,0) (Fig 2A  and 2B; Fig 3A and 3B). In C. asper, these processes are absent in the Indian Ocean population but present in the Western Atlantic population, and thus this terminal was coded as [1]. 8) Rostrum shape: character state 0 -rostrum reduced and not trough-shaped; character state 1 -developed and trough-shaped (CI = 100; RI = 100).
Rostral keel is a ventral expansion of the rostrum, located between the nasal capsules. Dalatias and Isistius lack rostral keel in the neurocranium (9,0) whereas in all other taxa examined this keel is present (9,1). 10) Rostral keel extension: character state 0 -short, never transcending anterior margin of nasal capsules; character state 1 -elongate, always transcending anterior margin of nasal capsules (CI = 100; RI = 100).
Rostral appendages may be present laterally and medially as hook-like projections for connecting anterolaterally the nasal capsules to the ventral base of the rostrum by ligaments. Dalatias and Isistius have no rostral appendages. These are also absent in species of Cirrhigaleus (11,0) but present in all Squalus species examined (11,1). 12) Number of rostral appendages: character state 0 -one pair of lateral rostral appendages; character state 1 -one pair of lateral rostral appendages plus one medial rostral appendage (CI = 100; RI = 100).
In Cirrhigaleus and Squalus, profundus canal has one ventral aperture placed in the interorbital wall and a second aperture that opens up to the ethmoidal region, called herein dorsal aperture (13,1) [22,63]. Stated that the profundus nerve runs together with superficial nerves in the preorbital canal in Dalatias and Isistius, thus, profundus canal is absent (13,0). 14) Position of the dorsal aperture of profundus canal: character state 0 -aside the ethmoidal canal; character state 1 -between ethmoidal canal and preorbital canal (CI = 50; RI = 75).
In S. acanthias, S. suckleyi, S. megalops, S. brevirostris, and S. albifrons profundus canal has its dorsal aperture placed aside the ethmoidal canal, at the base of the nasal capsule (14,0). In Cirrhigaleus and S. mitsukurii, S. montalbani, and S. japonicus the dorsal aperture is between the ethmoidal canal and the preorbital canal at the longitudinal sulcus (14,1). 15) Relative width between postorbital processes and nasal capsules of the neurocranium: character state 0 -width of the across postorbital processes greater than to nasal capsules; character state 1 -neurocranium equally wide across nasal capsules and postorbital processes; character state 2 -neurocranium with greatest width across nasal capsules (CI = 50; RI = 71).
Antorbital cartilage is an extension of the subethmoidal region that is triangular and directed posteriorly, forming the ventral base of the preorbital wall. It is vestigial in Dalatias and Isistius as well as in Cirrhigaleus (16,0). In Squalus species, the antorbital cartilage is well developed and expanded posteriorly (16,1) (Fig 4). No homology between this character and the one similarly described for non-squalean sharks and batoids in Shirai (1992) is implied in the present study as this requires further investigation. Character codification follows Shirai (1992Shirai ( , 1996.
Pectoral and pelvic skeleton.
Pectoral fins are supported internally by pectoral basal and radials that vary in shape and name across Squaliformes. Dalatias and Isistius have one single basal in the pectoral fin (17,1). Squalus and Cirrhigaleus have three separated pectoral fin basals (17.0) (Fig 6).
In elasmobranchs, pectoral articular regions between the pectoral basals of the pectoral fin and pectoral girdle vary in number of articular surfaces and the types (condyle or facet) of articular surfaces [32]. This character refers to the number of surfaces in the scapula for articulation between the pectoral basals and pectoral girdle. As Dalatias and Isistius have one pectoral basal the pectoral fins articulate through a single articular region (18,0) while Squalus and Cirrhigaleus have two separate articular regions (18,1). 19) Articular region of pectoral propterygium: character state 0 -condyle; character state 1facet (CI = 100; RI = 100).
A condyle for articulation with the pectoral propterygium is found in Cirrhigaleus species (19,0) (Fig 7). In Squalus, a facet articulates with the propterygium (19,1). Since Dalatias and Isistius present one single pectoral basal and thus not homologous to the condition observed in Cirrhigaleus and Squalus, this character and also character 20 were coded as inapplicable for both taxa. 20) Articular region of pectoral mesopterygium and metapterygium: character state 0 -one condyle for articulation with mesopterygium only; character state 1 -one condyle for articulation with both mesopterygium and metapterygium (CI = 100; RI = 100).
The depressor pectoralis fossa exhibits a smooth and cylindrical dorsal ridge in the scapula in Dalatias, Isistius and Cirrhigaleus (21,0) (Fig 7A-7F). Species of Squalus also show cylindrical ridge in this region with a few exceptions. Squalus acanthias (Fig 7G-7I) and S. suckleyi have scapula with segmented ridge comprised by small barrel-shaped units located lateral-dorsally to the depressor pectoralis fossa (21,1). These barrel-shaped units were observed in [64,65] for S. acanthias. [66] suggested that this character may be an autapomorphy for this species but our analysis shows that the barrel-shaped elements may or may not be present in S. suckleyi. 22) Articular region for the basipterygium: character state 0 -one facet articulating with the basipterygium; character state 1 -one facet and one condyle articulating with basipterygium (CI = 100; RI = 100).
Puboischiadic bar of the pelvic girdle articulates with the pelvic fin through two different regions, one articular region for the articulation with the anterior pelvic basal (= first enlarged radial as in [66] of the pelvic fin and a second one with the basipterygium of the pelvic fin. The type of surfaces for the articulation with the pelvic fin may vary between facet or condyle. In Dalatias and Isistius, puboischiadic bar has one pelvic condyle posterolaterally on each side articulating with the anterior pelvic basal, and an inner facet posterolaterally on each side and medially to the pelvic condyle for articulating with the basipterygium. Squalus species also exhibit this pattern of pelvic articulation (25,0) as noticed in [66]. Cirrhigaleus species have the same pattern of pelvic articulation between the puboischiadic bar and anterior pelvic basal but the articular region for the basipterygium has two articular surfaces, an inner facet and a condyle (25,1) (Fig 8A-8F). Pelvic facet is placed ventromedially and the condyle more dorsolaterally in relation to the facet. This condition was not noticed for C. barbifer in [66] possibly due to analysis limitations of the dissected specimen. 23) Number of pelvic foramina: character state 0 -one single foramen; character state 1 -two foramina (CI = 33; RI = 60).
In Isistius, posterior medial process is conspicuously elongate, extending backwards as a triangular projection (28,0). In Dalatias and Squalus species, posterior medial process is short, not extending backwards as a triangular projection (28,1
In claspers, the axial cartilage may be straight or exhibit some sinuousity and/or a mesial expansion at the proximal edge, herein called mesial process. In Dalatias and Isistius, mesial process is absent (29,0). Squalus megalops, S. brevirostris and S. albifrons share the same condition. Squalus acanthias, S. suckleyi, S. mitsukurii, S. montalbani, S. japonicus as well as species of Cirrhigaleus exhibit a conspicuous mesial process in the axial cartilage (29,1 Dorsal-fin spines may be present or not at the basal cartilage of the dorsal fin. These spines may be located internally thus retained underneath the skin or externally arising above the skin. In Dalatias and Isistius dorsal-fin spines are absent (33,0 Anterior margin of nostrils has nasal lobes that are supported internally by flesh or cartilage. In Dalatias and Isistius, nasal lobes are supported by flesh (35,0). In Cirrhigaleus, the same condition is observed. In Squalus, nasal lobes are supported internally by thin cartilages (35,1). 36) Number of nasal lobes at anterior margin of nostrils: character state 0 -two; character state 1 -one (CI = 100; RI = 100).
Anterior margin of nostrils may have one or more nasal lobes, one laterally and a second lobe medially. Dalatias and Isistius have a single nasal lobe (36,1). The same condition is observed for S. acanthias and S. suckleyi but a medial nasal lobe may be present in neonates. Cirrhigaleus barbifer ( Fig 11A) and C. australis ( Fig 11B) have nasal barbels conspicuously moustache-like and conspicuously elongate, with its tips reaching the mouth (37,1). In C. asper (Fig 11C), the nasal barbel is non moustache-like and very short, with its tips never reaching the mouth (37,0). Nasal barbels are absent in Squalus, Isistius and Dalatia and thus these terminal taxa were coded as "?" for this character.  Upper labial furrow length is much shorter than lower labial furrow in Cirrhigaleus species (39,0) whereas upper labial furrow is as elongate as the lower labial furrow in species of Squalus (39,1). 40) Dorsal fin shape: character state 0 -dorsal fins similar in shape; character state 1 -dorsal fins dissimilar in shape (CI = 100; RI = 100). Inner margin of dorsal fins may be similar in length to the dorsal fin height. This condition is present in Dalatias, Isistius, S. acanthias and S. suckleyi (42,0). Cirrhigaleus species and Squalus megalops, S. brevirostris, S. albifrons, S. mitsukurii, S. montalbani and S. japonicus have dorsal-fin inner margin much smaller than dorsal fin height (42,1). 43) Proportional length between pectoral-pelvic fins and pelvic-caudal fins: character state 0pectoral-pelvic space equal or greater pelvic-caudal space; character state 1 -pectoral-pelvic space smaller than pelvic-caudal space (CI = 50; RI = 80).
The caudal penduncle may exhibit an upper precaudal pit which is a small notch or concavity placed just prior the origin of the caudal fin. In Dalatias and Isistius, precaudal pits are absent (44,0). The same condition is observed for Cirrhigaleus. Species of Squalus have precaudal pits (44,1) as it was noticed in [22] for the genus. Dorsal caudal margin is usually more elongate than the ventral caudal margin which confer a rectangular shape to the caudal fin. Sometimes the ventral caudal lobe is as elongate as the dorsal caudal margin and thus the caudal fin is triangular in shape. Isistius has this pattern of caudal fin shape (46,1) whereas Dalatias, Cirrhigaleus and Squalus have rectangular caudal fin (46,0).
Cusplets are structures that are accessory to the main cusp and it is usually smaller in length. The cusplets are located medially to the lateral cusp on either side of the posterior margin of the crown base. In C. asper (Fig 12I-12L) these cusplets are inconspicuous (48,0) as in Squalus. In C. barbifer and C. australis (Fig 12A-12H) these cusplets are conspicuous (48,1). 49) Ridges on the crown surface: character state 0 -absent; character state 1 -present (CI = 100; RI = 100). Dermal denticles present medial and lateral ridges at the dorsal surface of the crown in most taxa examined (49,1). Dalatias and Isistius do not have medial and lateral ridges (49,0). 50) Number of ridges in the dermal denticles: character state 0 -three ridges; character state 1 -single ridge (CI = 100; RI = 100).

Morphological phylogenetic reconstruction
Maximum parsimony analysis performed herein included 51 morphological characters (four quantitative and 47 qualitative) and 13 terminal taxa. We chose the cladistic analysis using implied weighting with a concavity constant equal to 1 as the preferred hypothesis, considering its stronger support and greatest internal resolution of clades. This analysis resulted in two equally most-parsimonious trees with 69.9 steps, CI = 0.80 and RI = 0.88. The character matrix was divided into quantitative characters with absolute and normalized values (S1 Table) and qualitative characters (S2 Table) for each terminal analyzed. The list of synapomorphies presented below refers to the family Squalidae, Squalus and Cirrhigaleus. Relative Bremer support and GC values are shown in the cladogram (Fig 13) below each node. Morphological evidence that supports each node is summarized below.
Clade E (S. albifrons, S. brevirostris and S. megalops) is supported by two synapomorphies: mesial process absent in the axial cartilage of the claspers (ch. 29, 1>0) and ventral terminal cartilage greater than one-third the length of the axial cartilage (ch. 30, 1>0). Squalus albifrons is placed at the base of this clade and has no autapomorphies. Squalus brevirostris and S. megalops (Clade F) are hypothesized as closely related and this relationship is supported by lower values of monospondylous vertebrae (ch. 1, 0.375>0.250-0.313); no autapomorphies were found for both taxa. Clade G consisting of S. mitsukurii, S. japonicus and S. montalbani is supported by the presence of one pair of lateral rostral appendages plus one medial rostral appendage (ch. 12, 0>1). Squalus mitsukurii has no autapomorphies. A close relationship between S. japonicus and S. montalbani (Clade H) is hypothesized based on equally wide nasal capsules and postorbital processes (ch. 15, 0>1). Squalus japonicus is characterized by a ventral terminal cartilage shorter than one-third the length of axial cartilage (ch. 30, 1>0) whereas S. montalbani is characterized by a neurocranium with greatest width across nasal capsules (ch. 15, 1>2) and two intermediate segments between the basipterygium of the pelvic fin and the axial cartilage of the claspers (ch. 26, 0>1).
Clade I consists of all species of Cirrhigaleus and its monophyly is supported by eight synapomorphies: higher values of monospondylous vertebrae (ch. 1, 0.375>0.750); presence of an innervation of the medial nasal lobe by the buccopharyngeal branch of the facial nerve (ch. 5, 0>1); fleshy core present at the anterior margin of nostrils (ch. 6, 0>1); neurocranium with greatest width across nasal capsules (ch. 15, 1>2); one facet and one condyle for articulating with basipterygium of the pelvic fin (ch. 22, 0>1); two intermediate segments between the basipterygium and the axial cartilage of the claspers (ch. 26, 0>1); five terminal cartilages of the claspers (ch. 28, 1>2); posterior medial process of the puboischiadic bar absent (ch. 24, 0>1). Cirrhigaleus asper is hypothesized as sister-group of the clade comprising C. australis and C. barbifer (Clade J) and this species has no autapomorphies. Clade J is supported by the presence of conspicuous cusplets at the crown of dermal denticles (ch. 48, 0>1); C. australis and C. barbifer have no autapomorphies.

Systematics of Squalidae
Phylogenetic analysis of 51 morphological characters supports the monophyly of Squalidae which is congruent with previous hypothesis of [15,22]. Out of the 18 synapomorphies for the clade, only one was previously proposed: dignathic homodonty in [22]. Presence of a facet on the pectoral articular region for propterygium was proposed as synapomorphy of Squalidae in [32] and again in [49,51] which is in contrast to our current analysis. Cirrhigaleus share a condition similar to those observed for Etmopteridae (as described in [51]): a condyle for the propterygium and a condyle for the mesopterygium and metapterygium.
Some synapomorphies, however, require revision as other members within Squaliformes that were left out of the present analysis. Squalidae share the same conditions related to the number of pectoral basals with Etmopteridae and Echinorhinus [22,37,48,67], and the number of pectoral articular regions with Chlamydoselachidae [32] and Etmopteridae [51]. Other characters, including rostral keel in the neurocranium, and number of terminal cartilages on claspers, number of nasal lobes at nostrils, shape of dermal denticles and number of vertebrae have been overlooked for Squaliformes. A more inclusive cladistic analysis of morphological characters for representatives of all families within Squaliformes is required in order to revise the synapomorphies proposed here for Squalidae and test the influence of homoplastic characters. As the scope of this study is to elucidate the taxonomic classification of Cirrhigaleus and to test its inner phylogeny we will no further discuss the relationships of Squalidae and its monophyly within the order.

Monophyly of genera and inner relationships
Morphological phylogenetic reconstruction undertaken in this study supports the monophyly of Cirrhigaleus. The genus comprises a clade that is sister-group to a second clade, representing the genus Squalus. Thus, the current analysis supports the validity of Cirrhigaleus as suggested in [18,19,22] but refute the molecular hypothesis using NADH2 gene of [15] in reallocating Cirrhigaleus species into the genus Squalus (Fig 14).
For Cirrhigaleus, besides the synapomorphy raised in [22] that is the presence of an innervation of the medial nasal lobe by the buccopharyngeal branch of the facial nerve (ch. 5 in the present analysis) other seven novel synapomorphies are hypothesized in the current analysis. Cirrhigaleus is furthermore defined as a genus of roughskin dogfish sharks with higher values of monospondylous vertebrae counts, medial nasal lobe supported by fleshy core and innervated by the buccopharyngeal branch of the facial nerve, neurocranium with greatest width across nasal capsules, one facet and one condyle in the puboischiadic bar for articulating with the basipterygium, two intermediate segments between the basipterygium of the pelvic fin and the axial cartilage of the claspers, five terminal cartilages of the claspers, and posterior medial process of the puboischiadic bar absent [22]. Described the fleshy core of the nostrils in Cirrhigaleus but it did not take into account in his analysis. The pelvic articular region described here for the genus is proposed for the first time and it is in contrast to the recent description of [66] observed for C. barbifer. According to these authors, the puboischiadic bar articulates with the basipterygium through a single enlarged and reniform facet located on each posterolateral portion of the pelvic girdle and medially to the position of the condyle for articulation with the anterior pelvic element (sensu first enlarged radial in [66]). Cirrhigaleus species lack the posterior medial process of the puboischiadic bar (sensu posteromedian projection) as also noticed in [66]. Characters of the claspers (e.g., number of intermediate elements; number of terminal cartilages) are cladistically analyzed herein for the first time and should be taken into account in future phylogenetic analysis within Squaliformes. Descriptive anatomy of the claspers has been provided to discuss the interrelationships in sharks and batoids as seen in [68,69] and more recently [70,71]. [27,47] treated clasper morphology within Squaliformes but since then no additional examination has been conducted, except for general species descriptions such as for Isistius in [35,52]. The monophyly of Squalus is supported by rostral appendages present in the neurocranium, presence of paired supraethmoidal processes in the neurocranium, antorbital cartilage developed and expanded posteriorly, nasal lobes supported by cartilage, and presence of precaudal pits. With exception to the presence of precaudal pits proposed in [22], the characters raised here are all novel synapomorphies of the genus. Presence of supraethmoidal process in the ethmoidal region of the neurocranium is synapomorphic for the genus and thus in congruence with [22]. Plasticity of supraethmoidal processes in C. asper, however, have resulted it as uninformative character for Cirrhigaleus.
Furthermore, novel insights into interrelationships within Squalus are highlighted in the current analysis especially regarding the 'validity' of group/complex of species. In the resulting mostparsimonious morphological tree, all three groups of species of Squalus are monophyletic. Squalus acanthias group (Clade C) consisting of S. acanthias and S. suckleyi is sister-group of all other Squalus members. Squalus megalops group (Clade E), herein comprising of S. albifrons, S. megalops and S. brevirostris, is sister-group of the S. mitsukurii group (Clade G) consisting of S. mitsukurii, S. montalbani and S. japonicus. The topology provided here represents the first hypothesis of morphological interrelationships within the genus and it is partially congruent with the most recent molecular phylogenetic hypothesis (e.g. [15,72,73]) in supporting three separate clades within Squalus (Fig 14). Our analysis further provides a better resolution with regards to the validity of the complex/group of species as the inner and interrelationships between the S. mitsukurii and S. megalops groups have been thought to be unclear and unresolved. With exception to the S. acanthias group (Clade C) and a smaller clade consisting of S. megalops and S. brevirostris (Clade F) that exhibit Bremer support and GC value above 30%, other clades and phylogenetic arrangements between Squalus species reveal weak support possibly due to operational issues as only a few Squalus species were included in the analysis for outgroup inference. These results, however, are tentative as the focus of the current study is on Cirrhigaleus. Future investigations on the phylogenetic interrelationships within the genus Squalus may reveal a different scenario by increasing the number of characters and adding other terminal taxa to the analysis. Whether the validity of the complexes/groups of species is again supported the taxonomic classification for the whole genus must be revised. Since S. acanthias is the type-species of the genus, the clade comprising S. acanthias and S. suckleyi could retain the designation Squalus, and the remaining two clades should be given a new generic designation. Flakeus Whitley, 1939 [74] and Koinga Whitley, 1939 [74] are generic names currently in synonymy with Squalus and may be resurrected.

Generic placement of C. asper
Phylogenetic analysis of morphological characters presented here supports that the nominal species C. asper is correctly assigned to the genus Cirrhigaleus and that this species is sistergroup to a small clade consisting of C. barbifer and C. australis. These results are congruent to the evidence presented earlier in [3,6,22] that supported to be congeneric with other Cirrhigaleus species but against [2,8,15] on suggesting its generic allocation to Squalus. Cirrhigaleus barbifer and C. australis share a condition of the dermal denticles that is the presence of cusplets at the posterior margin of the crown which is absent in C. asper. This species thus has shown to share many meristics, inner and external morphological characteristics with Cirrhigaleus congeners that was not previously known including higher number of monospondylous vertebrae, medial nasal lobe (nasal barbels) supported by fleshy core and innervated by the buccopharyngeal branch of the facial nerve, neurocranium with greatest width across nasal capsules, pelvic articular region comprised by one facet and one condyle for articulating with the basipterygium, two intermediate segments between the basipterygium and the axial cartilage, and five terminal cartilages of the claspers.
External morphology, meristics and morphometric data of C. asper are much more similar to its congeners. It shares with C. barbifer and C. australis many characteristics, such as: body trihedral and markedly robust; dermal denticles tricuspid and conspicuously wide with cusplets; teeth unicuspid with apron markedly broad and cusp somewhat upright; first and second dorsal fins vertical and upright, equally tall; first and second dorsal-fin spines almost equal in length (second dorsal-fin spine often worn down but not broken); origin of second dorsal fin placed over a vertical line traced at pelvic-fin free rear tips; upper labial furrow markedly short with thick fold.
Morphological differences are more apparent between species of Cirrhigaleus and Squalus related to, for instance, upper labial furrow small with thick fold (vs. upper labial furrow large and thin in Squalus), spiracles above the eyes (vs. eyes placed laterally behind the eyes in Squalus), dermal denticles with cusplets at posterior margin of the crown (vs. cusplets absent in Squalus), and second dorsal fin with its origin over pelvic free rear tips (vs. origin of second dorsal fin far behind pelvic free rear tips in Squalus) [2,8]. Noticed morphological differences between C. asper and species of Squalus, that are also observed in this study, including: size of dermal denticles (three times larger in C. asper than in species of Squalidae); position of origin of first dorsal fin (behind free rear tips of pectoral fins in C. asper vs. prior or over free rear tips of pectoral fins in Squalus, except for S. acanthias); origin of pelvic fins (just prior origin of second dorsal fin in C. asper vs. conspicuously prior to second dorsal fin in Squalus); length of caudal peduncle (shorter in C. asper than in Squalus), and precaudal pit (absent in C. asper vs. present in Squalus). Similarities between Cirrhigaleus and Squalus regarding dentition, length of dorsal-fin spines, shape of dermal denticles, absence of nasal barbels, presence of precaudal keel, vertebral counts and morphology of terminal cartilages of the claspers led the misleading generic placement of C. asper and synonymy of Cirrhigaleus in [2,8] as many of the characteristics pointed out in these studies can be applied to other species of Cirrhigaleus as well as some members of Squaliformes (e.g. Centrophorus).
In particular, presence or absence of nasal barbels has been applied as diagnostic character to separate species of Squalus and Cirrhigaleus. Cirrhigaleus asper and species of Squalus bear anterior margin of nostrils conspicuously short and thus the former species have been misidentified as Squalus. Nostrils internal anatomy is not homologous within Squalidae as so a precautionary approach has to be conducted to diagnose species. Nasal barbels are here defined as in [22] as an extension of the mesial nasal lobe that is supported internally by fleshy core and innervated by a buccopharyngeal branch of facial nerve (VII). Nasal barbels differ in length within Cirrhigaleus as C. barbifer and C. australis exhibit conspicuously elongated and moustache-like nasal barbels whereas C. asper shows short and non moustache-like barbels. Species of Squalus do not bear nasal barbels because the mesial nasal lobe is supported internally by a thin nasal cartilage with not associated innervations. Additionally, lateral nasal lobe is often slightly larger than medial lobe in species of Squalus as stated previously in [4] while Cirrhigaleus species have lateral nasal lobe much shorter than medial one, including C. asper.
Type species: Cirrhigaleus barbifer Tanaka, 1912 [1] by original designation and monotypy. Diagnosis. A genus of the family Squalidae differing from Squalus by bearing nasal barbel in the anterior margin of nostrils that it is innervated by a branch buccopharyngeal of the facial nerve (VII) and internally supported by fleshy core. Cirrhigaleus is distinct from Squalus on lacking precaudal pits (vs. present in Squalus), spiracles placed dorsally above the eyes (vs. spiracles placed laterally behind the eyes), eyes with both anterior and posterior margins notched (vs. eyes with anterior margin concave and posterior margin notched), upper labial furrow very short with thick fold (vs. upper labial furrow large with thin fold), and origin of second dorsal fin over or just prior a vertical line traced at pelvic-fin free rear tips (vs. origin of second dorsal fin far behind a vertical line traced at pelvic-fin free rear tips). Cirrhigaleus also differs from Squalus by having dorsal-fin spines markedly elongate, transcending greatly dorsal-fin apexes (vs. dorsal-fin spine short, rarely transcending dorsal-fin apexes). Caudal fin of Cirrhigaleus exhibits continuous transition between upper and lower lobes at level of caudal fork, a condition distinct from Squalus (vs. discontinuous transition between upper and lower lobes). Cirrhigaleus body trihedral in cross-section, conspicuously arched dorsally and humped at belly, tail fusiform and short dorsal-caudal space (vs. body entirely fusiform from head to tail, inconspicuously arched dorsal and ventrally at belly, elongate dorsal-caudal space in Squalus). Cirrhigaleus species also show higher values of monospondylous vertebrae counts than Squalus species (47-53 for Cirrhigaleus vs. 37-50 for Squalus).
Geographical distribution. Cirrhigaleus occurs from the North to Southwestern Pacific Oceans, including waters off Japan, Indonesia, New Zealand and New Caledonia, and in the Western Indian Ocean from the Seychelles to South Africa and St. Paul Islands, as well as in the North and South Western Atlantic Oceans from the USA to Brazil (Fig 15).
Remarks. Three valid species of barbel-bearing dogfishes of the genus Cirrhigaleus are here recognized: C. barbifer, C. asper and C. australis. They comprise benthic species that are often found on or near bottom of the outer continental shelves and mid-continental slopes in depths between 91-1103 meters. According to [6,12], species are rarely caught off bays and river mouths. Tanaka Diagnosis. Cirrhigaleus barbifer is distinguished from its congeners by having body dark grey dorsally (vs. light grey for C. australis vs. brown for C. asper), and teeth with apron conspicuously broad (vs. narrower in C. australis and C. asper). It is easily distinct from C. asper by having nasal barbels moustache-like and well elongate (5.4%-6.4%TL), often reaching the mouth (vs. nasal barbel non moustache-like and markedly short, its length 1.0%-1.5% TL, slightly transcending posterior margin of nostrils in C. asper), and dermal denticles bat-like (vs. heart-shaped) with median ridge very narrow (vs. broad) and lateral cusps conspicuous (vs. inconspicuous). Cirrhigaleus barbifer is separated from C. australis by having more elongate nasal barbels (5.4%-6.4%TL vs. 4.4%-6.2%TL, respectively) and dermal denticles with a two cusplets on each side (vs. one cusplet in C. australis) (Fig 16).

Cirrhigaleus barbifer
Description. Morphometric and meristic data are shown in Table 1 and S3 and S4 Tables. Single value is for neotype and ranges values are for other material.
Dermal denticles. Dermal denticles tricuspid and bat-like, somewhat imbricate, large and conspicuously broad at the crown (Fig 12A-12C); dermal denticles with its width as large as its length; cusps conspicuous, pointed and located posteriorly at posterior margin of the crown; median cusp much more elongate than lateral ones; median ridge elongate, thin, tall and straight, transcending anteriorly the crown base; lateral ridges slender, short, low and conspicuously convex proximally; median furrow narrow and shallow, placed anteriorly over median ridge; lateral furrows short and profound, located anteriorly aside each lateral ridge; two secondary cusps (or cusplets) often present on each side of posterior margin of the crown; cusplets much smaller than median cusp.
Colouration. In fresh, body brownish grey at the dorsal and at the upper lateral half, whitish ventrally (Fig 16). Nasal barbels white, sometimes greyish lateral and ventrally at its distal end. Dorsal fins grey to light grey with somewhat dark grey submarginal bar; dorsal-fin posterior margins broadly white from apex to its middle line; dorsal-fin free rear tips whitish. Dorsal-fin spines greyish brown, whitish at the tips. Pectoral fins brownish grey dorsally and whitish ventrally with large dark grey blotches throughout the edges; pectoral-fin posterior margin slightly

PLOS ONE
Untangling the systematic dilemma behind Cirrhigaleus asper white. Pelvic fins brownish grey dorsally and white ventrally with large light grey blotches throughout the edges; pelvic-fin posterior margin slightly white. Caudal fin brownish grey throughout all its extension; preventral caudal margin white, with blackish subterminal stripe; postventral caudal margins narrowly white with dark grey subterminal bar internally; black caudal stripe evident anteriorly. In preserved specimens (Fig 20), body dark grey to brownish dorsal and laterally, white ventrally. Nasal barbels white, greyish lateral and ventrally in its distal end. Dorsal fins grey, somewhat light grey posteriorly in the submarginal bar; dorsal-fin posterior margins broadly white from apex to its middle line; dorsal-fin free rear tips whitish. Dorsal-fin spines dark brown, whitish at the tips. Pectoral fins dark grey dorsally and whitish ventrally with large light grey blotches; pectoral-fin posterior margin uniformly white. Pelvic fins grey dorsally and white ventrally with large light grey blotches; pelvic-fin posterior margin white. Caudal fin dark grey throughout all its extension; postventral caudal margins narrowly white with blackish subterminal bar internally; black caudal stripe evident anteriorly.
Geographical distribution. Cirrhigaleus barbifer occurs exclusively in the Pacific Ocean in waters from Japan, Indonesia, and New Caledonia as well as in Western Australia (Fig 15). It is caught between 200-760 meters depth.
Remarks. Designation of the neotype of C. barbifer. Tanaka (1912) [1] described both genus and species based in a single mature male collected at the Tokyo Fish Market in Japan that was designated originally as type and deposited under the catalogue number 3397 in the Science College Museum in Tokyo (currently ZUMT). It is possible that the holotype came from the fish collection of Mr. A. Owston because Dr. S. Tanaka used to work on specimens collected or donated by him as it is provided in [1,[90][91][92][93]. Some authors (e.g., [17,19]) made references to the illustration of the holotype provided in [1] rather than to the specimen itself. According to Dr. K. Sakamoto (ZUMT), the holotype is lost. No other specimen of C. barbifer exists at the ZUMT fish collection, according to verifications achieved personally by the first author of this manuscript, suggesting that the holotype no longer exists. Interestingly, there is no other specimen in the fish collections from Japan collected from the same locality as the type locality of C. barbifer, the Sagami Sea, provided in [1], which reject the possibility of the holotype to have been transferred to another collection in the past. A specimen of C. barbifer (SU 14171) that was also collected by Mr. A. Owston is available at CAS and it has same collecting data of those of the holotype of C. barbifer including collecting site and total length. However, this specimen is a mature female (not a mature male), according to D. Catania (CAS), which discards the possibility of being a missing holotype that ended up at CAS and received a new catalogue number by mistake on the accession and assignment of the specimen.
Cirrhigaleus barbifer has been regularly confused with C. australis in the South Pacific Ocean due to their morphological similarities regarding body size and shape, length of nasal barbels and dorsal-fin spines, and shape of dorsal and caudal fins as well as shape and size of dermal denticles. The characteristics provided in [1] for C. barbifer may also be applied to C. australis. [3] tentatively provided morphological diagnostic characters for separating these two nominal species but many of these (e.g., eye length, dorsal-caudal space, and vertebral counts) exhibited continuous range of values. These studies indicate clearly the difficulty to properly separate C. barbifer and C. australis. Moreover, instraspecific variations (ontogenetic and regional) observed here within C. barbifer reveal a more complex scenario behind the taxonomy of this nominal species because it obscures its morphological differentiation with congeners. Here, the neotype of C. barbifer (HUMZ 197852) is designated to clarify the taxonomic status of this species for better defining morphologically its local form and separating it effectively from C. australis. The neotype is held at Hokkaido University and it was collected off Kanaya, Gulf of Tokyo in Japan, which it is the locality nearest to the original type locality of C. barbifer. Following the requirements of International Code of Zoological Nomenclature (ICZN), diagnosis and description of this species are efficiently provided in our study, and the differentiation with closest related species (C. australis) are provided below based on characters of shape of dermal denticles, number of intermediate teeth, body colour, and length of nasal barbels.
Synonymy of C. barbifer. [84] described Phaenopogon Herre, 1935 [84] as a new genus and monotypic to include P. barbulifer Herre, 1935 [84]. This species was described based on a single specimen collected from Misaki Bay (Sagami Sea), Japan in 1906 by Mr. Alan Owston. This author did not take into account the previous study of [1] as he himself mentioned later in [76]. The characteristics provided in [84] are congruent with those observed for C. barbifer, including length and shape of nasal barbels, shape of dermal denticles, body colour, shape of fins and proportional external measurements. These observations indicate that P. barbulifer is junior synonym of C. barbifer, which is in agreement with [3,14,76].
Intraspecific variations. Origin of first dorsal fin is anterior or over a vertical line traced at pectoral-fin free rear tips in juveniles of C. barbifer while it is behind it in adults. First dorsalfin spine length reaches up to half of first dorsal-fin height and never transcends first dorsalfin apex in juveniles while first dorsal-fin spine length is over half of first dorsal-fin height and conspicuously transcends first-dorsal fin apex in adults. Second dorsal-fin spine is also smaller in juveniles (7.1%-7.5% TL) than in adults (7.6%-8.0% TL), reaching or mostly often transcending its second dorsal-fin apex in adults but not so often in juveniles. Nasal barbels exceed posterior margin of mouth in juveniles while in adults it reaches anterior margin of mouth or just before it, which it is in agreement with [4]. Height of first and second dorsal fins increases with growth (first dorsal-fin height 8.9%-10.0% TL in juveniles vs. 10.0%-10.8% TL in adults; second dorsal-fin height 8.8%-9.6% TL vs. 9.3%-10.5% TL, respectively). Other measurements that vary ontogenetically in the Japanese species are: pre-second dorsal length (60.8%-61.0% in juveniles vs. 62.0%-66.5% TL in adults); pre-vent length (52.5%-54.8% TL in juveniles vs. 54.4%-56.3% TL in adults; prespiracular length (9.5%-9.8% TL in juveniles vs. 9.7%-11.3% TL in adults); length of pelvic fin (12.2%-13.3% TL in juveniles vs. 13.1%-14.4% TL in adults).
Dorsal fins conspicuously upright and vertical (Fig 23), both equally tall with first dorsal fin height 1.0 (0.9-2.1) times height of second dorsal fin; first dorsal fin as large as second dorsal fin, its length 1.0 (0.9-1.5) times length of second dorsal fin. Origin of the first dorsal fin located behind the vertical traced at pectoral free rear tips, although near to it in most specimens. First dorsal fin with anterior margin convex and large, its length 13.7% (10.8%-14.5%) TL; first dorsal-fin posterior margin straight and large, its length 10.1% (6.9%-11.8%) TL; first
Pectoral fin conspicuously wide distally and square-like ( Fig 24A); pectoral-fin anterior margin convex and elongate, its length 1.7 (1.2-1.9) times larger than pectoral-fin inner margin length, and corresponding at least to one-half the distance between pectoral and pelvic fins; pectoral-fin inner margin markedly convex; pectoral-fin posterior margin straight and large, its length 10.5% (8.7%-12.9%) TL; apex and free rear tip of pectoral fins conspicuously rounded and broad, not lobe-like and almost reaching the same length. Pectoral-pelvic distance 1.1 (0.7-1.5) times pelvic-caudal space; the latter 1.0 (0.8-1.3) times interdorsal space. Pelvic fins in the midline between first and second dorsal fin, although nearest to second dorsal fin in some paratypes and non-type specimens. Pelvic fins markedly broad with anterior margin somewhat convex (Fig 24B), posterior margin straight; pelvic-fin apex rounded and conspicuously wide, and free rear tips rounded and lobulated, although more slender in males.
Claspers in adults slender and flattened ventrally (Fig 25); claspers very short its inner length 1.1, (0.4-1.2) times pelvic inner margin length, transcending pelvic free rear tips; clasper groove very large, vertical, placed dorsally; apopyle and hypopyle with narrow apertures, Caudal peduncle very short with dorsal-caudal space 9.5% (7.4%-10.8%) TL with lateral keel prominent and thick since insertion of second dorsal fin to behind origin of caudal fin; upper and lower precaudal pits absent. Caudal fin (Fig 24C) rather rectangular with dorsal caudal margin convex anteriorly and turning straight until the dorsal caudal tip; length of dorsal caudal margin 0.9 (0.8-1.0) times head length and 1.7 (1.7-3.2) times larger than length of preventral caudal margin; dorsal caudal tip pointed; upper post-ventral caudal margin concave, although straight near the dorsal caudal tip; lower post-ventral caudal margin straight; preventral caudal margin convex and short, its length 1.8 (1.4-3.1) times larger than inner length of pelvic fin; caudal fin slightly continuous between lobes and markedly broad at fork, its width 7.8% (6.8%-13.0%) TL.
Dermal denticles. Dermal denticles tricuspid and heart-shaped (Fig 12I-12L), conspicuously broad at the crown and imbricated; denticles with its width as large as its length; median cusp prominent, pointed and elongate; lateral cusps very short to inconspicuous; median ridge straight, tall, short, markedly thick anteriorly and thin posteriorly; conspicuous anterior furrow and often two small ridges on each side; median ridge projecting anteriorly beyond the crown base; one or two lateral ridges on each side, straight to slightly convex, tall and short, reaching the tip of crown edge; lateral furrow very profound and wide, placed aside each lateral ridge.
Colouration. In fresh material (Fig 26), body light grey dorsally and laterally up to the origin of pectoral fin, light grey laterally, and white ventrally. Pectoral and pelvic fins grey dorsally and whitish ventrally with light grey blotches at the edges and posterior margins white. Dorsal fins light grey and dark grey at the base of the dorsal-fin spines; dorsal-fin posterior margin broadly white from apex to free rear tips; submarginal bar broadly black. Dorsal-fin spines brownish grey, whitish at the tip. Caudal fin light grey, whitish at the vertebral column region; postventral caudal margins white with ventral caudal tip broadly white; upper caudal blotch broadly black; lower subterminal bar broadly black at the lower caudal lobe. In preserved specimens, body brownish-grey dorsally and white ventrally; pectoral, pelvic, dorsal and caudal fins darker than the rest of the body. Pectoral fins with inner and posterior margins homogenously white. Pelvic fins pale ventrally with inner and posterior margins broadly white. Dorsal fins with posterior margins broadly white from apex to free rear tips; dorsal fins lighter at the fin base near the dorsal-fin spine. Dorsal-fin spines brown, whitish at its base and tip. Caudal fin also brown with white postventral caudal margins, except at the dorsal caudal tip; ventral caudal tip broadly white; dorsal caudal margin white, except near the dorsal caudal tip; ventral caudal lobe with white basal marking; caudal stripe light brown.
Geographical distribution. It occurs in the Western Indian Ocean (Seychelles to South Africa and south to St. Paul Islands) and Western Atlantic Ocean (Texas, USA to Rio de Janeiro, Brazil), including the Caribbean Sea (Venezuela [108] and Mexico [110]). Cirrhigaleus asper is often caught between 91-750 meters depth. Its occurrence in Hawaii, USA is not confirmed as material from this region was not assessed in the present study (Fig 15).

Remarks
Intraspecific variations. Regional variations in morphometrics are observed in C. asper when only adult specimens are compared, especially between populations of the Western Central Atlantic Ocean (WCAO) and the other two populations in the Western Indian Ocean (WIO) and South-Western Atlantic Ocean (SWAO). For instance, prenarial length (4.2%-5.0% TL for WIO vs. 2.9%-3.1% TL for WCAO vs. 3.6%-4.6% TL for SWAO). WCAO also exhibit the greatest variations, regarding internarial width, upper labial furrow length, length of first dorsal-fin posterior margin, length of first dorsal-fin spine, height of second dorsal fin, and length of dorsal caudal margin. These specimens also have greater range of values than the WIO specimens for length of preventral caudal margin, pectoral-fin base length, and length of first dorsal-fin inner margin. SWAO specimens vary from those of the WCAO on first dorsal-fin length, length of second dorsal-fin posterior margin, pectoral-fin posterior margin length, and caudal fork width.
This species also exhibits apparent ontogenetic variations in external morphometrics within the WIO population. Changes with growth are observed for precaudal length, pre-second dorsal length, pre-spiracular length, preoral length, distance between nostrils and upper labial furrow, eye length, length of first dorsal fin base, dorsal caudal margin length and clasper length, and length of the nasal barbels. These variations, however, are not observed in the SWAO population and it is unknown for the WCAO population as the current study did not examine young specimens collected from this region. elongate, its length 4.4%-6.2% TL, usually reaching the mouth (vs. nasal barbel non moustache-like and conspicuously short, its length 1.0%-1.5% TL, never reaching the mouth in C. asper), and dermal denticles bat-like with narrow median ridge (vs. heart-shaped with broad median ridge in C. asper). It further differs from C. asper by smaller length of upper labial furrow, its length 0.9%-1.4% TL (vs. 1.4%-2.1% TL). Cirrhigaleus australis is separated from C. barbifer by having teeth with narrow apron (vs. broad in C. barbifer), and dermal denticles with one cusplet lateral-posteriorly on each side (vs. two cusplets in C. barbifer) (Fig 27).
Dermal denticles. Dermal denticles (Fig 12D-12H) tricuspid and bat-like, imbricate, large and conspicuously broad at the crown; dermal denticles with its width equivalent to its length; cusps pointed and located posteriorly at the crown margin; median cusp much larger than lateral ones; median ridge elongate, thin, tall and straight, slightly transcending anterior base of crown; lateral ridges thin, short and low, markedly convex proximally; median furrow small and shallow, placed proximally over median ridge; lateral furrow short and deep, located aside each lateral ridge; one secondary cusp (or cusplet) sometimes present on each side of posterior margin of the crown; cusplets much smaller than median cusp.
Colouration. Body light grey dorsal and laterally, white ventrally (Fig 27). Nasal barbels white, and slightly grey lateral-proximally. First dorsal fin dark grey with posterior margin broadly white from its apex to free rear tip, submarginal bar light grey, white spots in the anterior margin near its apex. Second dorsal fin dark grey with posterior margin broadly white until its midline, and submarginal bar light grey. Dorsal-fin spines white, greyish proximal and distally, although whitish at the tips. Pectoral fins grey dorsally and light grey ventrally, with both inner and posterior margins discreetly whitish at the edge. Pelvic fins also grey, lighter ventrally, with posterior margin sparsely whitish. Caudal fin dark grey, whitish medially; postventral caudal margins white but not uniform, broadly white in the dorsal and ventral caudal tips; subterminal bar blackish; black caudal stripe absent. In recent collected specimens, large dark grey blotches are observed ventrally in both pectoral and pelvic fins.
Geographical distribution. Cirrhigaleus australis occurs in the South-western Pacific Ocean and it is endemic to New Zealand and Southern Australia from Tweed Heads in New South Wales to Tasmania. It is caught between 91-1103 meters depth, and more often between 540-580 meters (Fig 15).
Remarks. Intraspecific variations. Snout and nasal barbels are smaller in adults than in juveniles of C. australis from Australia (AUS) but not in the New Zealand (NZ) population (prenarial length 3.9%-4.2% TL for adults vs. 4.5%-4.9% TL for juveniles; length of anterior margin of nostrils 4.4%-5.3% TL for adults vs. 5.9%-7.1% TL for juveniles). Clasper length also varies with maturity in both populations (clasper outer length 1.2%-1.5% TL for juveniles vs. 4.4% in adults for AUS; 3.7%-10.0% and 1.2%-1.7% for NZ, respectively; clasper inner length 3.5%-3.9% TL in juveniles vs. 7.2% TL in adults for AUS; 4.3%-8.8% and 3.2%-3.6% for NZ). Other variations are observed here for the first time, including position of origin of first dorsal fin varies from over or just anterior to the vertical traced at the pectoral-fin free rear tips in juveniles while it is behind it in adults.
C. australis exhibits morphometric and morphological variations between the New Zealand (NZ) and Australian (AUS) populations, such as: head longer and flattened dorsally in the NZ specimens (vs. shorter and arched in the AUS specimens); snout more obtuse (vs. snout rounded); dorsal fins broad at the fin web with straight posterior margin (vs. dorsal fins thin at fin web with strongly concave posterior margins); second dorsal-fin spine transcending second dorsal-fin apex (vs. not transcending second dorsal-fin apex); narrower abdomen when adults (7.3%-12.6% TL for NZ vs. 13.1%-16.1% TL for AUS, respectively).
It is also noticed that specimens from New Zealand exhibit intermediary characteristics, concerning body colour, dentition and shape of dermal denticles, shape of dorsal fins, length of dorsal-fin spines related to dorsal-fin apex, and origin of first dorsal fin related to pectoral-fin free rear tips, suggesting that more than one morphological group of Cirrhigaleus occurs in this region. One group has greyish body, origin of first dorsal fin prior to a vertical line traced at pectoral-fin free rear tips, first dorsal-fin spine never reaching first dorsal-fin apex, and caudal fin with posterior caudal tip narrow. The second group has dark brown body, origin of first dorsal fin behind a vertical line traced at pectoral-fin free rear tips, first dorsal-fin spine transcending first dorsal-fin apex, and caudal fin with posterior caudal tip broad. These specimens also have greater range of values of external measurements (e.g., precaudal length; pre-first and pre-second dorsal length; interdorsal space; dorsal-caudal distance; pectoral-pelvic distance; pelvic-caudal distance; size of first and second dorsal fins; length of dorsal-fin spines) and vertebral counts, which give additional support for this hypothesis.

Internal morphological description of Cirrhigaleus species
Neurocranium (Table 4) (Fig 2A and 2B; Fig 4A and 4B). In C. barbifer, neurocranium very thick, broader anteriorly across nasal capsules than posteriorly across postorbital processes (width across nasal capsules 68.2% CL vs. width across postorbital processes 62.2% CL in C. barbifer; 62.5%-70.5% CL and 55.1%-71.9% CL in C. asper; 68.0% and 59.7% CL in C. australis); neurocranium narrower in the interorbital region (its width 37.5% CL in C. barbifer, 30.6%-34.3% CL in C. asper and 33.1% CL in C. australis) and posteriorly in the occipital region (width across opistotic processes 41.6% CL in C. barbifer, 39.8%-43.6% CL in C. asper and 43.0% CL in C. australis). Precerebral fossa very deep distally, and shallow proximally. Prefrontal fontanelle markedly narrow. Rostrum spoon-like and very short (its length 39.9% CL in C. barbifer, 29.0%-34.1% CL in C. asper and 38.7% CL in C. australis), uniform on its extension with somewhat cylindrical lateral cartilages, rounded at the tip; rostral keel prominent, although markedly short (its length 22.6% CL), not reaching the anterior margin of the nasal capsules. Nasal capsules conspicuously large, oval dorsally and rounded ventrally, slightly oblique; many large nasal foramina evident lateral and ventrally in each nasal capsule, rarely in dorsal view; nasal capsule cracks present ventrally. Subnasal fenestra oval, slender and small on each side of the rostral keel at the base of prefrontal fontanelle. Ethmoidal region strongly narrow; epiphysial pit large and rounded, located anterior-dorsally just posteriorly to the prefrontal fontanelle with thick anterior crest. Ectethmoidal chamber markedly narrow ventrally, and antorbital cartilage tapered, continuous and convex at its distal margin. Subethmoidal region conspicuously short and slender, its width 15.2% CL in C. barbifer, 17.0%-18.4% CL in C. asper and 17.8% CL in C. australis); subethmoidal ridge prominent.
Cranial roof rather flattened with superficial longitudinal sulcus, carrying 10 tiny foramina of the branch superficial ophthalmic of the trigeminal and facial nerves (V-VII) plus preorbital canal; the latter conspicuously large and rounded, placed in front of the series of foramina for the ophthalmic nerve (VII); ethmoidal canal large, located well anteriorly in the base of the nasal capsule; profundus canal for the deep ophthalmic of the trigeminal nerve (V) with two apertures, one dorsal, very small and rounded, and another lateral, small and rounded, located in the interorbital wall just before the preorbital canal; dorsal aperture of the profundus canal placed between the ethmoidal canal and preorbital canal. Interorbital region concave with supraorbital crest slender and C-shaped; distance between orbital processes very small, its length 34.0% CL in C. barbifer, 36.0%-43.8% CL in C. asper and 32.2% CL in C. australis); preorbital process inconspicuous; postorbital process triangular and small, its length 13.4% CL in C. barbifer, 11.2%-14.4% CL in C. asper and 10.0% CL in C. australis); width across postorbital processes slightly greater than the width across preorbital processes (the former 62.2% CL and the latter 60.8% CL in C. barbifer, 55.1%-71.9% CL and 57.2%-64.5% CL in C. asper, and 59.7% and 53.5% CL in C. australis).
Preorbital wall convex and short. Interorbital wall profound with eye stalk small and wide, carrying distal disc, and located more posteriorly; foramen optic (II) very large and rounded, placed more ventral-anteriorly near the preorbital wall; foramen trochlear (IV) dorsally near the series of foramina of the branch superficial ophthalmic of the trigeminal nerve (V-VII); foramen oculomotor (III) just above the eye stalk while foramen abducens (VI) is near its base; trigeminal (V) and facial (VII) nerves with a common aperture, the foramen prooticum, posteriorly in the orbital wall; foramen prooticum also opens lateral-posteriorly to a branch hyomandibular of facial nerve (VII) in the hyomandibular facet through two small apertures. Basal angle prominent (its width 25.5% CL in C. barbifer, 17.8%-29.4% CL in C. asper and 18.8% CL in C. australis), located ventral-posteriorly in the interorbital wall for supporting the orbital articulation; foramen for an efferent of the pseudobranchial artery small and rounded, placed dorsally in the basal angle.
Otic region with endolymphatic fossa hexagonal and profound, carrying two anterior endolymphatic foramina and two posterior perilymphatic foramina, with similar sizes; otic capsule pentagonal and wide on each side; anterior semicircular canal inconspicuous; posterior and lateral semicircular canals well prominent on each otic capsule; the latter placed laterally in the otic capsule above the hyomandibular facet; otic crest very small and prominent, placed posteriorly between endolymphatic fossa and foramen magnum; sphenopterotic ridge subtle; opisthotic process somewhat pointed; hyomandibular facet shallow; lateral auditory groove markedly deep in the anterior edge of the lateral otic wall; inconspicuous lateral commissure.
Occipital region with occipital condyles small and triangular, ventrally; foramen magnum broad (its width 7.9% CL in C. barbifer, 7.6%-15.5% CL in C. asper and 4.1% CL in C. australis); foramen for vagus nerve (X) also large aside the foramen magnum; glossopharyngeal base broad, thick, and subtriangular, carrying glossopharyngeal foramen (IX) with two apertures; one aperture rounded and large placed posterior-dorsally, and a second aperture small and oval, located lateral-ventrally. Basal plate flattened and elongate (its length 51.5% CL in C. barbifer, 44.0%-50.3% CL in C. asper and 41.6% CL in C. australis), narrower anteriorly (its width 25.0% CL in C. barbifer, 21.8%-30.5% CL in C. asper and 41.6% CL in C. australis) than posteriorly in the glossopharyngeal base; basitrabecular processes been-shaped, prominent and very narrow, directed lateral-posteriorly; a single cartilaginous process, short and rounded, placed laterally in each side of the subotic shelf, width across cartilaginous processes 36.0% CL in C. barbifer, 35.4%-42.8% CL in C. asper and 33.5% CL in C. australis; a single foramen for carotid artery, rounded, placed mesial-anteriorly between basitrabecular processes and first cartilaginous processes; foramen for orbital artery, oval and small, located laterally near the base of each cartilaginous process.
Cirrhigaleus asper and C. australis have neurocranium of morphological condition similar to that of C. barbifer. Cirrhigaleus asper, however, differs from these species by having foramen for carotid artery with two apertures (vs. a single aperture for C. barbifer and C. australis), anterior semicircular canal prominent (vs. inconspicuous for C. barbifer and C. australis), and hyomandibular facet profound (vs. very shallow for C. barbifer and C. australis). Foramina of the branch superficial ophthalmic of the trigeminal nerve (V-VII) varies from 8-11 in C. asper while in C. australis varies from 7-9 foramina. C. asper exhibits intraspecific variation of the supraethmoidal processes, a paired short and cylindrical process located at the dorsoposterior edge of the prefrontal fontanelle. These processes may be absent (Indian Ocean population, including the holotype of C. asper) or present (Western Atlantic population) within the species. The supraethmoidal processes are also absent in both C. barbifer and C. australis. Cirrhigaleus barbifer and C. australis also have epiphyseal pit large and rounded with single and independent aperture, and it is not fused to the dorsal base of the prefrontal fontanelle as it was illustrated in [22]. Cirrhigaleus asper differs from C. barbifer and C. australis by: shorter precerebral fenestra length ( Pectoral apparatus. Pectoral girdle (Fig 6) constituted by a ventral transverse element, the coracoid bar, and a pair of scapula that is continuous to the coracoid bar, ending into a pointed tip, the scapular processes. Coracoid bar together with the scapula forms the scapulocoracoid cartilage. Scapulocoracoid is U-shaped, somewhat sinuous and cylindrical in general morphology, and placed transversely in relation to the body axis with scapular processes directed dorsal-posteriorly. Coracoid bar (Fig 7A-7F) straight and horizontal with its anterior margin conspicuously pointed and triangular, forming a prominent dorsal fossa for the insertion of the hypobranchial longitudinal muscles; ventral posterior margin of coracoid bar conspicuously pointed and convex on its midline; subrectangular and narrow prominence observed lateral-ventrally on each side of the coracoid bar. Posteriorly, a pair of lateral fossa supports the origin of the parietalis pars epaxonica muscle on the coracoid bar; a robust and triangular process, also called caudal process or posterior process of coracoid bar, is evident on the hindmost part of each fossa that together with the base of the articular process supports the origin of pterygii ventralis muscle. Scapula placed more dorsal-posteriorly, very broad ventrally and tapering cylindrically to its dorsal extremities where it turns into a pointed scapular process; its anterior margin markedly convex and posterior margin concave; there is a conspicuous and expanded lateral-anterior fossa on each side of scapula for origin of the pterygii cranialis muscle; this anterior fossa also carries a large and rounded foramen for the pectoral fin nerve, the foramen diazonale; foramen diazonale with small posterior aperture, placed more laterally between the two articular regions; foramen for pectoral artery located laterally just above the mesocondyle; scapula with prominent posterior ridge, placed more dorsally near scapular process. Scapular process somewhat cylindrical, tapering to its dorsal tip and directed medially to the body horizontal axis; scapular process attached proximally to the scapular process by thick connective tissue. Posteriorly, the pterygii dorsalis fossa, placed more distally above the articular region of the pectoral girdle, supports the origin of the pterygii dorsalis muscle in the scapula.
Propterygium divided into two pieces, one proximal, well elongate and conical, slenderer distally, and one distal, rectangular and cylindrical, shorter than the proximal one; propterygium carries one series of segmented radials in Cirrhigaleus barbifer. Mesopterygium triangular, much broader distally than proximally, carrying 8 series of segmented radials. Metapterygium triangular, thin, and elongated, narrower than the mesopterygium, carrying 8 series of segmented radials; metapterygium axis with a rectangular element, carrying one series of radials and another lateral and subtriangular element with three small radials attached to it. Pectoral radials segmented into three elements as well; distal radials longer than the proximal and mesial radials in propterygium and mesopterygium, while in metapterygium is shorter than the proximal and mesial radials; first series of radials in the mesopterygium is segmented into two elements only.
Cirrhigaleus asper and C. australis share similar morphology of the pectoral apparatus of that of C. barbifer. Cirrhigaleus australis and C. asper bear same number of pectoral radials for each pectoral element as those of C. barbifer. Articular region between pectoral girdle and fin comprises by two distinct regions in C. asper and C. australis, the procondyle and a mesometacondyle. Procondyle elliptical, oblique and well prominent, located lateral-dorsally in the scapula base, articulating with the propterygium; meso-metacondyle rounded and very large, placed posterior-ventrally in the scapula base with two distinct areas for articulating with the mesopterygium and metapterygium; the largest area articulates with the mesopterygium and it is located more lateral-posteriorly in the scapula; the second area is smaller and located more posterior-medially in the scapula for articulation with the metapterygium.
Pelvic apparatus. Pelvic girdle (Fig 8A-8F) with pubosichiadic bar transverse, large and narrow with anterior and posterior margins straight, although slightly convex medially; two small foramina for pelvic nerves evident laterally in the pubosichiadic bar, although anterior-most foramen is located right in the edge of lateral prepelvic process. Pelvic fin with anterior pelvic basal (= first enlarged radial) subtriangular, small and conspicuously broad with two series of radials associated to it (first radial is markedly broad and segmented); basipterygium elongate, cylindrical and thin, slightly broader on its proximal end than on its distal end; pelvic radials large, cylindrical and segmented into proximal and distal elements, the former much larger than the distal elements; 16-18 total pelvic radials for C. barbifer and 18 total pelvic radials for C. australis. Females of C. barbifer, C. asper and C. australis have one intermediate segment, small, barrel-shaped, attached proximally to the distal end of basipterygium, and a modified pelvic radial attached to the intermediate segment. In C. barbifer and C. asper this latter cartilage is rectangular, wide, bifurcated and cylindrical distally while in C. australis is ray-like, thin and cylindrical. These three species of Cirrhigaleus have two regions of pelvic articulation, one lateral and one medial. First region consists of a latero-posterior pelvic condyle in the puboischiadic bar that is prominent, rounded and elongate. It articulates laterally the puboischiadic bar to the anterior pelvic basal of the pelvic fin. Second articulating region is located at the medial posterior margin of puboischiadic bar and is comprised by two articular surfaces, one condyle and a pelvic facet. Pelvic facet is flattened and rectangular, placed ventromedially for articulating medially to the basipterygium. The second condyle is smaller than first condyle and it is located more dorsolaterally than the facet. The second condyle articulates to the dorsal portion of the basipterygium. The articular surface in the basipterygium is comprised by a small and rounded hollow for articulating with the second condyle.
Cirrhigaleus asper and C. australis differ from C. barbifer by having anterior foramen for pelvic nerve not completely enclosed in the lateral prepelvic process. This foramen is placed more posteriorly in C. australis while is more anteriorly in C. asper. Anterior pelvic basal of pelvic fin is rectangular and much larger in C. asper and C. australis than in C. barbifer. Four series of radials thin, cylindrical and segmented (except first one) are shown in C. asper while C. australis and C. barbifer exhibit two series of radials broad in which first series is wider than second one. Cirrhigaleus asper differs from C. barbifer and C. australis by having puboischiadic bar with anterior margin conspicuously convex medially, and posterior margin concave with paired convexity medially.
Cartilages of the claspers. Claspers (Fig 10) with two intermediate segments, barrel-shaped and thick connecting basipterygium to axial cartilage. First intermediate segment of C. barbifer is smaller than second one. Cirrhigaleus australis and C. asper apparently also have two intermediate segments that articulate basipterygium of the pelvic fin in females. Beta cartilage in C. barbifer is conspicuously stout and placed laterally over distal edge of basipterygium, first and second intermediate segments and proximal edge of axial cartilage transcending its mesial process. Axial cartilage almost straight and very elongate (its length equal to length of basipterygium of pelvic fin) with mesial process. Dorsal marginal cartilage strongly thick, although short (its length less than one-half length of axial cartilage), placed laterally over axial cartilage. Dorsal terminal cartilage thick, somewhat blade-like and elongate (its length one-half length of axial cartilage), located distally and connect proximally to dorsal marginal cartilage and axial cartilage. Dorsal terminal 2 cartilage leaf-like and conspicuously slender, although wider proximally, and large (its length one-third greater than length of dorsal terminal cartilage, although not reaching tip of dorsal terminal cartilage), connected proximal-medially to dorsal marginal cartilage and medial-distally to dorsal terminal 2 cartilage. Ventral marginal cartilage subtriangular and conspicuously broad distally, very elongate (it transcends midline of axial cartilage), attached to distal edge of axial cartilage; ventral marginal cartilage has folded plate markedly profound in which accessory terminal 3 cartilage is inserted. Accessory terminal 3 cartilage blade-like and heavy, although somewhat slender and cylindrical distally. Ventral terminal cartilage spatula-like, convex at its tip and laterally, markedly large (its length twice length of ventral marginal cartilage), attached proximally to ventral marginal cartilage. Accessory ventral marginal 2 cartilage present, located over proximal edge of ventral terminal cartilage, rectangular in shape and elongate (its length one-half length of ventral terminal cartilage).

Taxonomic considerations within Cirrhigaleus
Three species of barbel-bearing dogfish sharks are recognized in this study with occurrences in limited deep-sea regions of all oceans: C. barbifer, C. australis and C. asper. Cirrhigaleus barbifer occurs from North to South Pacific Ocean between Japan, Taiwan, New Caledonia, Western Australia and Indonesia as noticed in [1, 3-5, 7, 86]. This species is also reported in Vanuatu [5] and Taiwan [87] but examination of specimen vouchers and/or genetic analysis were not assessed in the present study to corroborate these assumptions. Cirrhigaleus australis is endemic to Southern Australia and New Zealand waters in the Pacific Ocean as suggested in [3,82] but in contrast to [12] for the Indian Ocean. Cirrhigaleus asper occurs in the Western Indian and North to South Western Atlantic Oceans as noticed in [2,8,12,83,99,100,101,114]. Its occurrence in the Hawaiian Islands and in the Central Pacific Ocean [88,103,107] is still doubtful.
Cirrhigaleus species are easily differentiated by characteristics of external morphology as provided in their diagnosis and key to species. Dermal denticles are tricuspid and conspicuously broad with cusplets on each side in C. barbifer and C. australis, which it is in disagreement with [3] who only noticed this character for the Australian species. Cirrhigaleus barbifer, however, differs from C. australis in number of cusplets (two cusplets vs. one cusplet, respectively). Cirrhigaleus asper lacks cusplets. Other characters of the dermal denticles that help to separate these species are its shape (bat-like in C. barbifer and C. australis vs. heart-shaped in C. asper), width of median ridge (narrow in C. barbifer and C. australis vs. broad in C. asper), and lateral cusp (conspicuous in C. barbifer and C. australis vs. inconspicuous in C. asper).
Dental formula of C. barbifer is congruent with C. asper and C. australis, although the some specimens of this species may present one intermediate tooth on upper and lower jaws (vs. absence of intermediate tooth). Dentition differentiates C. barbifer from congeners regarding width of apron that is much broader in this species. Body colouration is also helpful for taxonomic separation, which is in agreement with [3,7] for C. barbifer and C. australis. These authors reported differences related to second dorsal-fin spine length, length of preventral caudal margin, eye length, and length of upper labial furrow between C. australis and C. barbifer, which are not supported in our study [3]. Further separated these species based on length of first dorsal-fin spine, second dorsal fin base length, height of second dorsal fin, prenarial length, interdorsal space, and length of pectoral-fin posterior margin, although these differences are not observed here. Vertebral counts overlap between C. barbifer and C. australis and it is consistent with observations of [3,4,7].
Morphometric characters are useful for separating species of Cirrhigaleus but it is more apparent when only adult specimens are compared due to strong intraspecific and ontogenetic variations. Growth changes are not consistent to all species of Cirrhigaleus, revealing that their taxonomy and morphology are much more complex. For instance, length of anterior margin of nostrils and length of claspers vary with growth in C. australis and C. asper while these are constant in C. barbifer. Pre-second dorsal length and prespiracular length vary from juveniles to adults in C. barbifer and C. asper while these characters are continuous in C. australis. Cirrhigaleus barbifer vary with growth regarding pre-vent length, prepelvic length, pectoral-pelvic space, length of first and second dorsal-fin inner margins, height of first and second dorsal fins, length of second dorsal-fin posterior margin, length of second dorsal-fin spine, length of pectoral-fin anterior margin, and length of pelvic fin. These variations are not observed for C. australis and C. asper. In contrast to C. barbifer and C. asper, C. australis exhibits ontogenetic variations in prenarial length. Precaudal length, distance between nostril and upper labial furrow, eye length, base length of first dorsal-fin, and dorsal caudal margin length vary in C. asper while these characters are constant in C. barbifer and C. australis. [3,4] previously reported morphometric growth changes in C. barbifer and C. australis, respectively. The former authors took into account measurements provided in [84] for the holotype of P. barbulifer to represent a juvenile female with 555 mm TL. Examination of this specimen reveals that its total length corresponds to 730 mm TL (and not 555 mm TL) and that this information has been erroneously reproduced elsewhere since [84] which possibly interfered in the results of [4] regarding growth changes. Despite of it, we support that height of first and second dorsal fins, and length of pectoral-fin anterior margin vary ontogenetically in C. barbifer as in [4] but these characters increase with growth which is in contrast to their study. Variations in preoral length, eye length, and length of dorsal caudal margin for C. barbifer are not supported in the current study, which is incongruent with [4]. [3] noticed variations in pre-branchial length, prepelvic length, space between pelvic and caudal fins, distance from pectoral to pelvic fins, eye length, interorbital space, and length of second dorsal spine between juveniles and adults of C. australis, which is in disagreement with the current analysis. Ontogenetic variations are more expressive in C. barbifer than in C. asper and C. australis. Morphological analysis further reveals intraspecific regional variations for all Cirrhigaleus species as described under their remarks, which contribute to the taxonomical complexity of the group that was previously speculative in [3,12,88]. Existence of possible separate morphotypes of C. barbifer in Indonesia/New Caledonia/Vanuatu/Taiwan, and C. asper in the USA and Brazil should be investigated using an integrative taxonomic approach by incorporating molecular markers and morphological characters of newly collected material. sending material on loan for dissection. Thanks to A. Paterson (SAIAB) for institutional support of this project. Special thanks also to F. Tashiro (HUMZ) and C. Struthers (NMNZ) for sending photos and data of specimen vouchers, and P. Roque and F. Hazin (UFRPE) for providing photos and collecting data of fresh specimens. We acknowledge that opinions, findings and conclusions or recommendations expressed in this publication, partly supported by NRF funds, are those of the authors, and that the NRF accepts no liability whatsoever in this regard.